L-Glutamate is a major excitatory neurotransmitter in the central nervous system (CNS), which acts through the ligand-gated ionotropic glutamate receptor or through the G-protein coupled receptors (GPCR) called metabotropic glutamate receptors (mGluR). The mGluR5 belongs to the Group I subclass and is coupled to the phosphoinositide/Ca+2 pathway, which mainly mediates the excitatory effects of glutamate. Recently, investigation into the role of mGluR5 in drug abuse has led to speculation that this may be a new target for medication development. For example, studies using either an mGluR5 antagonist or mGluR5 knockout mice showed reduced locomotor stimulant effects induced by cocaine. Moreover, evidence that mGluR5 is involved in the rewarding effects of morphine, nicotine and ethanol has also been reported. Thus development of selective mGluR5 antagonists may provide a novel non-dopaminergic strategy toward the discovery of drug abuse medications. Additionally the mGluR5 has recently been implicated in anxiety and depression thus these antagonists might provide new therapeutic agents toward the treatment of these CNS disorders. In order to further explore structure-activity relationships (SAR) at mGluR5, the design and synthesis of a series of diaryl amides was initiated based on a putative ligand binding site at the transmembrane domain region of an mGluR5 molecular model, based on the bovine rhodopsin crystal structure. In vitro binding and functional evaluation at mGluR5 resulted in the discovery of several novel and moderately high affinity mGluR5 antagonists. Additional modifications of these amide-linked molecules focused on inducing an intramolecular hydrogen bond that might provide co-planarity achieved in the parent compound MPEP. Within this new series of compounds, several analogues showed moderate affinity for mGluR5, but a better design in which co-planarity could be achieved through a quinoline group was pursued. In this first group of quinoline analogues, clues to improve binding affinity at mGluR5 were revealed. In addition, synthesis of additional alkyne and amide analogues have been achieved to further explore SAR of the pendant aryl ring and then to combine these features with the quinoline structure. A large series (>250 compounds) have now been evaluated for in vitro binding and function at mGluR5 and several candidates are being evaluated in animal models of anxiety. In addition, we have discovered that although the alkyne series has provided important guidance in our drug design of the amide and quinoline series, there are significant differences in which substitution is well-tolerated in the alkyne series but not in the other two templates. Recently we have developed a customized mGluR binding assay using 3HMPEP in rat brain, a calcium fluorescence functional assay in mGluR5 transfected HEK 293 cells and several in vivo models of anxiety, a hallmark behavior associated with mGluR5 antagonism, as well as models of addiction. This work is directed to understanding the role of mGluR5 in anxiety, drug abuse and addiction and to develop potential therapeutic agents for treatment. Nevertheless, it is known that mGluR5 heterodimerizes in cells, with dopamine (DA) D2 and A2A receptors and these heterodimeric complexes may be involved in the behaviors elicited by these agents. Hence, we plan to investigate the mGluR5-D2 heteromer and further, attempt to discover if mGluR5 also forms heteromers with dopamine D3 receptors. In order to elucidate mechanistic underpinnings of the potential therapeutic effects of these mGluR5 antagonists, we propose to develop cell lines that are transfected with these heteromeric complexes. We plan to screen the 200+ compounds generated in our lab, compare these data to the data obtained in the mGluR5 transfected cells and 1) draw conclusions of overlapping structure-activity relationships (SAR) or 2) observe significant differences in SAR suggesting that these complexes produce binding domains that differ from the homomeric receptor and might be a target for new drug discovery. Finally, correlation with in vivo behavior depending on what the result of these in vitro tests will be a future goal of this project.